SUMMARY Enhanced glycolysis and suppression of mitochondrial metabolism characterize the Warburg phenomenon in cancer cells. Metabolites enter and exit mitochondria through one channel in the outer membrane: the voltage dependent anion channel (VDAC). The central hypothesis of this proposal is that high free tubulin levels in cancer cells blocks VDAC and suppresses oxidative phosphorylation in Warburg metabolism and that reversal of tubulin inhibition of VDAC has an anti-Warburg effect that enhances oxidative phosphorylation, promotes oxidative stress and decreases glycolysis. We further hypothesize that small molecules antagonists of the inhibitory effect of VDAC on tubulin hyperpolarize mitochondria and increase generation of reactive oxygen species (ROS), leading to mitochondrial dysfunction and cell death. Accordingly in Specific Aim 1, we will characterize the effects of erastin and other VDAC-tubulin antagonists on cellular bioenergetics (ATP, ADP, AMP, Pi, NADH redox state, AMP kinase, and rates of respiration and glycolysis) in HepG2, Huh7 and FOCUS human hepatocarcinoma (HCC) cells. We will also assess in a Huh7 mouse xenograft model the effect of erastin/VDAC-tubulin antagonists on mitochondrial membrane potential (??) and the glycolytic phenotype. Lead compounds identified in a high throughput screening will be confirmed by electrophysiology as VDAC-tubulin antagonists, evaluated for effects on cellular bioenergetics and used to create a pharmacophore. In Specific Aim 2, we will assess the effects of protein kinase A (PKA)-dependent VDAC phosphorylation on the bioenergetic of HCC cells. We will use agonists and inhibitors of PKA as well as PKA overexpression and siRNA silencing in the presence and absence of erastin/VDAC-tubulin antagonists and after VDAC isoform double knockdown. Additionally, proteomic analysis will determine specific sites of VDAC isoform phosphorylation. In Specific Aim 3, we will determine the cytotoxic mechanisms of VDAC-tubulin antagonists. We expect that erastin and lead compounds will increase ?? and ROS formation, leading to the mitochondrial permeability transition, bioenergetic failure, and cell death. We will also determine if cell death occurs by apoptosis, necrosis or necroptosis and if mitochondrial function can be preserved by antioxidants. Overall, the project will generate fundamental new knowledge on mechanisms causing suppression of mitochondrial metabolism in HCC and will identify new agents that block VDAC-tubulin interaction to revert the pro-proliferative Warburg metabolic phenotype and selectively promote cytotoxic oxidative stress.